Xerostomia (dry mouth) is the most common complication of radiotherapy (RT) in head and neck squamous cell carcinoma (HNSCC). It adversely impacts patients' quality of life and places them at risk for significant late morbidities. Current strategies to mitigate xerostomia are costly and ineffective. Intensity modulated radiotherapy (IMRT), which aims to spare one parotid gland, has resulted in some improvement of stimulatory salivary function, but it cannot spare the submandibular glands (SMG), which are crucial for resting salivary function throughout the day, because of their location adjacent to the draining lymph nodes. Recently, adult stem cells (SC) have been isolated from murine SMG and have shown the ability to rescue salivary function in irradiated mice. We found that these SCs express a significantly higher level of Aldehyde Dehydrogenase-3A2 (ALDH3A2) than their non-stem-cell counterparts (NSCs) and treatment with an ALDH3A2 activator, Alda89, resulted in SMG SC enrichment in vivo. We also found that SMG irradiation resulted in hypoxia, which has been reported to decrease the overall ALDHS expression in normal cells. Hence, we hypothesize that RT-induced hypoxia in the SMG decreases the survival and regeneration of SMG SCs through its effect on ALHD3A2 expression, and activation of this enzyme either genetically or pharmacologically during or after RT may mitigate RT-induce xerostomia. Hypoxia signaling through the hypoxia inducible factor-1 (HIF-1) is critical for normal tissue homeostasis during RT-related inflammation. HIF-1 levels are regulated by the prolyl hydroxylases (PHDs), whose inhibition results in protection against RT-induced gastrointestinal toxicity and increased survival of lethally irradiated mice (Project 1). We hypothesize that HIF-1 signaling is also critical for the mitigation of RTinduced damage of the SMG and modulation of its activity with a PHD inhibitor will mitigate such damage. The main objectives of this proposal are: (1) to evaluate the effect of RT and hypoxia on ALDH3A2 expression and function in SMG SC and NSC, (2) to determine whether ALDHSA2 activation or PHD inhibition can mitigate RT-induced xerostomia (Project 1) and (3) to determine the effect of ALDH3A2 activation and PHD inhibition on HNSCC growth and survival (Project 1,3 and Core B). Our ultimate goal is to devise novel strategies to mitigate RT-induced xerostomia in HNC patients.